Molecular roulette: nucleophosmin mutations in AML are orchestrated through N-nucleotide addition by TdT.

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML). AML with mutated NPM1 is recognized as a separate entity in the World Health Organization 2016 classification and carries a relatively favorable prognosis. NPM1 mutations are predominantly 4-bp duplications or insertions in the terminal exon that arise through an unknown mechanism. Here we analyze 2430 NPM1 mutations from 2329 adult and 101 pediatric patients to address their origin. We show that NPM1 mutations display the hallmarks of replication slippage, but lack suitable germline microhomology available for priming. Insertion mutations display G/C-rich N-nucleotide tracts, with a significant bias toward polypurine and polypyrimidine stacking (P < .001). These features suggest terminal deoxynucleotidyl transferase (TdT) primes replication slippage through N-nucleotide addition, with longer syntheses manifesting as N-regions. The recurrent type A, type D, and type B mutations require 1, 2, and 3 N-nucleotide extensions of T, CC, and CAT, respectively, with the last nucleotide used as occult microhomology. This TdT-mutator model successfully predicts the relative incidence of the 256 potential 4-bp insertion/duplication mutations at position c.863_864 over 4 orders of magnitude (ρ = 0.484, P < .0001). Children have a different NPM1 mutation spectrum to adults, including a shift away from type A mutations and toward longer N-regions, consistent with higher TdT activity in pediatric myeloid stem cells. These findings complement our FLT3-ITD data, suggesting illegitimate TdT activity contributes to around one-half of AMLs. AML may therefore reflect the price for adaptive immunity.

Terminal deoxynucleotidyl transferase promotes acute myeloid leukemia by priming FLT3-ITD replication slippage.

FLT3-internal tandem duplications (FLT3-ITDs) are prognostic driver mutations found in acute myeloid leukemia (AML). Although these short duplications occur in 25% of AML patients, little is known about the molecular mechanism underlying their formation. Understanding the origin of FLT3-ITDs would advance our understanding of the genesis of AML. We analyzed the sequence and molecular anatomy of 300 FLT3-ITDs to address this issue, including 114 ITDs with additional nucleotides of unknown origin located between the 2 copies of the repeat. We observed anatomy consistent with replication slippage, but could only identify the germline microhomology (1-6 bp) anticipated to prime such slippage in one-third of FLT3-ITDs. We explain the paradox of the "missing" microhomology in the majority of FLT3-ITDs through occult microhomology: specifically, by priming through use of nontemplated nucleotides (N-nucleotides) added by terminal deoxynucleotidyl transferase (TdT). We suggest that TdT-mediated nucleotide addition in excess of that required for priming creates N-regions at the duplication junctions, explaining the additional nucleotides observed at this position. FLT3-ITD N-regions have a G/C content (66.9%), dinucleotide composition (P < .001), and length characteristics consistent with synthesis by TdT. AML types with high TdT show an increased incidence of FLT3-ITDs (M0; P = .0017). These results point to an unexpected role for the lymphoid enzyme TdT in priming FLT3-ITDs. Although the physiological role of TdT is to increase antigenic diversity through N-nucleotide addition during V(D)J recombination of IG/TCR genes, here we propose that illegitimate TdT activity makes a significant contribution to the genesis of AML.

NUP98-NSD1 fusion in association with FLT3-ITD mutation identifies a prognostically relevant subgroup of pediatric acute myeloid leukemia patients suitable for monitoring by real time quantitative PCR.

The cytogenetically cryptic t(5;11)(q35;p15) leading to the NUP98-NSD1 fusion is a rare but recurrent gene rearrangement recently reported to identify a group of young AML patients with poor prognosis. We used reverse transcription polymerase chain reaction (PCR) to screen retrospectively diagnostic samples from 54 unselected pediatric AML patients and designed a real time quantitative PCR assay to track individual patient response to treatment. Four positive cases (7%) were identified; three arising de novo and one therapy related AML. All had intermediate risk cytogenetic markers and a concurrent FLT3-ITD but lacked NPM1 and CEBPA mutations. The patients had a poor response to therapy and all proceeded to hematopoietic stem cell transplant. These data lend support to the adoption of screening for NUP98-NSD1 in pediatric AML without otherwise favorable genetic markers. The role of quantitative PCR is also highlighted as a potential tool for managing NUP98-NSD1 positive patients post-treatment.

1H MRS identifies specific metabolite profiles associated with MYCN-amplified and non-amplified tumour subtypes of neuroblastoma cell lines.

Neuroblastoma is the most common extracranial solid malignancy in children. The disease possesses a broad range of clinical phenotypes with widely varying prognoses. Numerous studies have sought to identify the associated genetic abnormalities in the tumour, resulting in the identification of useful prognostic markers. In particular, the presence of multiple copies of the MYCN oncogene (referred to as MYCN amplification) has been found to confer a poor prognosis. However, the molecular pathways involved are as yet poorly defined. Metabolite profiles generated by in vitro (1)H MRS provide a means of investigating the downstream metabolic consequences of genetic alterations and can identify potential targets for new agents. Thirteen neuroblastoma cell lines possessing multiple genetic alterations were investigated; seven were MYCN amplified and six MYCN non-amplified. In vitro magic angle spinning (1)H MRS was performed on cell suspensions, and the spectra analysed to obtain metabolite concentration ratios relative to total choline (tCho). A principal component analysis using these concentration ratios showed that MYCN-amplified and non-amplified cell lines form separate classes according to their metabolite profiles. Phosphocholine/tCho and taurine/tCho were found to be significantly raised (p < 0.05) and glycerophosphocholine/tCho significantly reduced (p < 0.05) in the MYCN-amplified compared with the MYCN non-amplified cell lines (two-tailed t test). (1)H MRS of the SH-EP1 cell line and an isogenic cell line transfected with the MYCN oncogene also showed that MYCN oncogene over-expression causes alterations in phosphocholine, glycerophosphocholine and taurine concentrations. Molecular pathways of choline and taurine metabolism are potential targets for new agents tailored to MYCN-amplified neuroblastoma.